Dipartimento di Chimica "Ugo Schiff", DICUS, Università di Firenze, Via della Lastruccia 3-13, I-50019 Sesto Fiorentino, Italy.
Department of Chemistry, Institute of Biochemistry, University of Natural Resources and Life Sciences, Muthgasse 18, A-1190 Vienna, Austria.
Biomolecules. 2023 Jan 25;13(2):235. doi: 10.3390/biom13020235.
Monoderm bacteria accumulate heme via the coproporphyrin-dependent biosynthesis pathway. In the final step, in the presence of two molecules of HO, the propionate groups of coproheme at positions 2 and 4 are decarboxylated to form vinyl groups by coproheme decarboxylase (ChdC), in a stepwise process. Decarboxylation of propionate 2 produces an intermediate that rotates by 90° inside the protein pocket, bringing propionate 4 near the catalytic tyrosine, to allow the second decarboxylation step. The active site of ChdCs is stabilized by an extensive H-bond network involving water molecules, specific amino acid residues, and the propionate groups of the porphyrin. To evaluate the role of these H-bonds in the pocket stability and enzyme functionality, we characterized, via resonance Raman and electronic absorption spectroscopies, single and double mutants of the actinobacterial pathogen ChdC complexed with coproheme and heme . The selective elimination of the H-bond interactions between propionates 2, 4, 6, and 7 and the polar residues of the pocket allowed us to establish the role of each H-bond in the catalytic reaction and to follow the changes in the interactions from the substrate to the product.
单胞菌通过粪卟啉原依赖的生物合成途径积累血红素。在最后一步，在两个 HO 分子的存在下，通过粪卟啉原脱羧酶（ChdC），以逐步的方式，将位置 2 和 4 的粪卟啉原的丙酸基团脱羧形成乙烯基基团。丙酸 2 的脱羧作用产生一种中间体，在蛋白质口袋内旋转 90°，使丙酸 4 靠近催化酪氨酸，从而允许进行第二步脱羧反应。ChdC 的活性位点通过涉及水分子、特定氨基酸残基和卟啉的丙酸基团的广泛氢键网络稳定。为了评估这些口袋稳定性和酶功能中的氢键的作用，我们通过共振拉曼和电子吸收光谱学，对与粪卟啉和血红素复合的放线菌病原体 ChdC 的单突变体和双突变体进行了表征。选择性消除丙酸 2、4、6 和 7 与口袋的极性残基之间的氢键相互作用，使我们能够确定每个氢键在催化反应中的作用，并跟踪从底物到产物的相互作用的变化。
